Internal death programs play significant roles in many diseases. Pathogenic effects can result from inefficient cell death or from inappropriate or excessive death such as that caused by the human immunodeficiency virus (HIV) during AIDS or the SAR-CoV virus during SARS. In this project, we are taking a multifaceted approach to studying molecular mechanisms of both apoptotic and nonapoptotic death programs in lymphocytes as well as other cell types. A major focus of our investigations are death-inducing cell surface receptors in the tumor necrosis factor receptor (TNFR) superfamily such as TNFR1 and CD95/Fas/APO-1. Both receptors play an important role in stimulating both apoptotic and nonapoptotic death of cells principally in immune processes. Little is known about how these alternative death pathways are entrained to receptor signaling. Interestingly, both receptors can have effects beside death such as the induction of transcription factors. We are trying to understand how these receptors stimulate the intracellular machinery that causes cell death in preference to other cellular outcomes. We have devoted many of our efforts to understanding the activation of a protease called caspase-8 which regulates the death program. We have characterized two death programs that emanate from TNFR1 and the Fas receptor, one which is caspase-8 dependent and has an apoptotic morphology and the other which is caspase-8 independent and involves necrosis. Interestingly, the latter death program is only observed when caspase-8 is inhibited. The regulation and molecular pathways of these two forms of lymphocyte death are distinct. In addition, we have discovered that inhibition of caspase-8 in non-lymphoid cells can lead to another form of cell death exhibiting particular cytoplasmic double membrane structures called autophagy. Although initially controversial, several labs have now shown that this form of death is particularly important for the demise of tumor cells by chemotherapeutic agents. We have now shown that the mechanism of autophagic death program is selective degradation of catalase which leads to a marked overaccumulation of reactive oxygen species leading to cellular damage and death. Furthermore, we have focused on genes that play key roles in this process of death. We have found that the human homologue of the Drosophila spinster protein, called hSpin, is essential for autophagic cell death. We have studied the biochemical function of this protein and found that it is important for proper lysosome biogenesis and vesicle trafficking. In particular, it plays a vital role in lysosomal resomation at the end of autophagy. These studies will shed light on nonapoptotic death mechanisms. In parallel, we are exploring how the regulation of cellular death programs may play a role in cytopathicity associated with virus infections in AIDS and SARS. In particular, a critical effect in the onset of AIDS following infection with HIV is the death of T lymphocytes caused by the virus. We have found that this death process is necrotic rather than apoptotic and have now identified two viral gene products, vif and vpr, that are involved in this process. We have found that vpr alters the cell cycle and promote death by binding to cellular proteins that have a role in cell cycle progression. In order to study this process rigorously we have constructed a mathematical model to analyze cell death in tissue culture during HIV infection. Remarkably, both of these cytotoxic gene products cause says cycle arrest at the boundary of the G2 and M phases. The mathematical model reveals that the principal cause of cell loss is cell death rather than cell cycle arrest. We are using molecular genetic approaches to determine if cell cycle arrest actually causes cell death and how this might come about. The HIV vpr protein is a small protein (100 amino acids) with no obvious structural domains or enzymatic motifs other than three alpha helices. We have determined that vpr promotes the formation of an apparently abortive complex between mitotic regulators such as CyclinB and Cdk1, and the theta isoform of the 14-3-3 protein which inhibits the cell cycle in the G2 phase. The complex appears to be nucleated by a particular hydrophobic patch on the third helix of the vpr protein. We have also studied how vif causes cell cycle arrest and found that it is a distinctive mechanism from that induced by vpr. We find that vif can alter the nucleocytoplasmic localization of cyclins and cyclin-dependent kinases which leads to disruption of normal cell cycle progression. We continue to explore how HIV-1 alters to cellular machinery to cause the demise of CD4 T cells. In contrast to HIV, the human coronavirus that causes SARS, SARS-CoV, causes necrotic cell death that does not involve cell cycle arrest. We have found that cell death can be traced to a novel open reading frame, termed ORF 3a, that is present in SARS-CoV but not other less pathological human coronaviruses. The cellular effect of ORF3a is to cause a dramatic reorganization of the Golgi apparatus which has lethal effects on the cell. We are now trying to established a molecular pathway entrained to ORF3a that causes this cytopathic effect. Thus far, our data implicate the TGN38 protein that is primarily found in the trans portion of the Golgi as the main direct target of the ORF3a protein. Further experiments will be directed at understanding the mechanism of how SARS triggers a lethal event through the alteration of the Golgi apparatus. Our explorations have revealed that the SARS virus causes cell death by a unique process involving Golgi apparatus disintegration which is also seen in nonlymphoid cells infected by other cytopathic viruses. The ORF3a encoded by SARS-CoV directly provokes Golgi disintegration. Understanding the molecular mechanism of this process may lead to valuable insights into viral cytopathicity. Also, SARS-CoV induced cell death might involve the ORF-3b protein. In examining ORFb, which, along with ORF3a is unique to SARS-CoV and not found in less pathogenic human coronaviruses, we found that it exhibited a novel form of temporal translocation of the protein between the mitochondria and the cell nucleus. Preliminary evidence suggests that it has an inimical effect on the normal interferon response against viral infection and we are currently exploring the molecular basis of this effect.